High throughput stem cell assay for identifying stem cells useful for transplantation

ABSTRACT

The present invention relates generally to high-throughput assay methods that determine the proliferative status of hematopoietic stem and progenitor cells. The present invention further relates to high-throughput assays for screening compounds that modulate the growth of hematopoietic stem and progenitor cells and for identifying subpopulations thereof that are suitable for transplantation. The assay of the present invention is particularly useful for quality control and monitoring of the growth potential in the stem cell transplant setting and would provide improved control over the reconstitution phase of transplanted cells.

PRIORITY CLAIM

This application claims priority as a divisional application under 35U.S.C. §120 to U.S. patent application Ser. No. 10/059,521, filed onJan. 29, 2002, now U.S. Pat. No. 7,354,729, which claims the benefit ofU.S. Provisional Patent Application Ser. No. 60/264,796, filed on Jan.29, 2001. The contents of each of these applications is herebyincorporated by reference in its entirety.

This invention was made with government support under Small BusinessInnovation Research (SBIR) grant number 1R43CA93244-02 awarded by theNIH. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to high-throughput assay methodsthat determine the proliferative status of hematopoietic stem andprogenitor cells. The present invention further relates tohigh-throughput assays for screening compounds that modulate theproliferation of hematopoietic stem and progenitor cells and foridentifying subpopulations thereof that are suitable fortransplantation.

BACKGROUND

Two major concerns in drug development are the need to predict theefficacy and safety of a potential drug candidate before clinical trialsare initiated, and how to predict which individual cancer cases aregoing to respond to a particular treatment. During the developmentprocess, therefore, drug candidates are typically batch tested on avariety of cell lines. Those that seem to have the desired effect on thecell lines are then tested in animals during the preclinical phase.Neither process, however, replicates the true clinical situation. As aresult, patients in clinical trials often have to endure unpleasant andsometimes harmful effects because neither differential sensitivity norpatient variability was adequately considered during the drugdevelopmental phases.

To overcome these deficiencies, in vitro cell-based assays thatdemonstrate a high degree of predictability during different phases ofdrug development are needed. In addition, there is a need for highthroughput assays for screening large numbers of potential drugcandidates produced during the discovery phase of drug development andallow those demonstrating promise to continue further in research anddevelopment. It therefore follows that assays based on primary cellsexhibiting a high degree of predictability, coupled with ahigh-throughput component, could have a significant impact and value onthe drug development process.

The hematopoietic system is one of five continuously proliferatingsystems of the body, the others being the epithelial mucosa of thegastrointestinal tract, the dermis of the skin, the germ cells of thereproductive organs and the epithelium of the eye cornea. All fiveproliferating systems share common characteristics, the most importantbeing that a small population of stem cells maintains the continuousproduction of mature end cells. They all possess the same structuralorganization of four basic compartments, namely the stem cell,amplification and differentiation, maturation and mature cellcompartments.

The hematopoietic system, however, is unique in several ways. It is theonly system capable of producing at least eight functionally differentcell lineages from a single pluripotent stem cell. Assays are availablethat allow the differential effect of drugs on the variouslympho-hematopoietic lineages to be examined. Second, the site of cellproduction changes during ontological development. This helps indifferential sensitivity testing. Third, the site of production in theadult is the bone marrow, which is a significantly different tissue fromthe functional site of the peripheral circulation. Fourth, compared withother proliferating systems, and almost all other systems of the body,adult hematopoietic stem and progenitor cells are readily accessible.

Hematopoietic stem and progenator cell lineages can be used to measureparameters that would normally be inaccessible. For example, thefunctional site of hematopoiesis is the circulation and mature end cellscan be readily obtained to measure red and white blood cell counts,differential counts and other end stage blood parameters. Theseparameters are conventionally used in preclinical drug testing and formthe basis of the National Cancer Institute (NCI) guidelines forhemotoxicity testing during clinical trials. However, these parametershave little if any predictive value as to, for example, the cytotoxiceffect of therapeutic compounds on primitive hematopoietic cells or thestem cells of other proliferating tissues.

Besides the mature red and white blood cells, the peripheral blood alsocontains circulating populations of stem and progenitor cells that canbe isolated and used for hematopoietic status monitoring andhemotoxicity testing. The so-called granulocyte-macrophagecolony-forming cell (GM-CFC) assay and the enumeration of CD34⁺ cells(stem and early progenitor cells) currently form the basis of qualitycontrol for hematopoietic stem cell transplantation.

The widespread use of in vitro hematopoietic assays was initiated whensoluble factors released by fibroblasts were shown to be capable ofstimulating cells to form granulocyte-macrophage colonies in soft agar(Bradley & Metcalf, Aust. J. Exp. Biol. Med. 44, 287-287 (1987); Pluznik& Sachs, Exp. Cell Res. 43, 553-553 (1966)). Colony forming assays(CFAs) for erythropoietic progenitor cells (McLeod et al., Blood 44,617-534 (1974); Iscove et al., J. Cell Phyisol. 2-23 (1974); Axelrad etal., Haemopiesis in Culture 226-223 (1974)) and other hematopoieticlineages were also developed. The use of cytotoxic drugs such as5-fluorouracil (Hodgson et al., Exp. Hemat. 10, 26-36 (1982) and Int. J.Cell Cloning 1, 49-56 (1983)) and hydroxyurea (Rosendaal et al., Nature264, 68-69 (1976)) allowed the hierarchy within the stem cellcompartment to be elucidated and in vitro assays for primitive stem cellpopulations to be developed (Ploemacher et al., Blood 78, 2527-2536(1991); Sutherland et al., Blood 72, 104a (1988)).

In vivo, an insult at the stem or early progenitor cell level requires acertain amount of time for the effect to be detected at the peripheralblood level. The effect may not be observed for weeks, or even months.This does not provide a high level of predictability and is why endstage cell parameters cannot be used to predict the effect of an agent.By the time the effect is observed, adverse reactions by the patienthave already occurred.

In vitro colony-forming assays based on stem or progenitor cells, on theother hand, can fulfill the requirements of prediction and sensitivitybecause they detect the effect of the insult before it is observed inthe circulation. Colony-forming assays for leukemic cells are alsoavailable. In these classic assays, the more primitive the cell to bedetected, the longer it takes to detect its progeny in the form of acolony. The proliferative potential of the cells being analyzed, andtheir ability to be stimulated by growth factors in vitro are essentialfor these assays. This dependency on the amplification compartmentinherent in the hematopoietic system is often overlooked and withoutthis component colony-forming assays in general, and especiallypredictive hemotoxicity testing, could not be performed.

Under steady-state conditions, the proliferative status of primitivestem cells is considered to be quiescent, while the proportion of cellsin cell cycle increases with stem cell maturity. Once the stem cell hasbecome determined with respect to a cell lineage, it enters theamplification compartment for producing the large and constant number ofmature cells. With entry into the cell cycle, however, the cell becomesvulnerable to exogenous agents including the cytotoxic drugs typicallyused in oncology. Thus, the GM-CFC assay, for example, has been used topredict myelosuppression (Prieto, P., Sci. Total Environ. 247, 349-354(2000)). The predictive quality of this assay, has been proven byvalidation studies with alkylating agents (Parchment et al., Toxicol.Pathol. 21, 241-250 (1993)). Additionally, however, if the maximumtolerated drug concentration for hematopoietic cells can be predicted,hemotoxicity studies would play an important role in drug discoverysince it would be a therapeutic index-based assay (Parchment et al.,Ann. Oncol. 9, 357-364 (1998)).

In the case of cytotoxic drug testing, the target cells have to be incell cycle. For any drug that relies on cell proliferation, the tissuesmost affected or damaged by toxicity are those actively engaged in cellproliferation, which includes the bone marrow and the gastrointestinaltract. It therefore follows that hemotoxicity testing could alsousefully be extrapolated to, and predictive for, the effects of apotential drug on other proliferating tissues.

Toxicity in general, and hemotoxicity in particular, can also becorrelated with the time of drug administration. The therapeutic indexof a drug, and hence its toxicity, is dependent, in part on thecircadian variation in the hematopoietic cell division of rodents(Laerum, O. D., Exp. Hematol. 23, 1145-1147 (1995); Aardal et al., Exp.Hemtol., 11, 792-801 (1993); Aardal, Exp. Hematol. 12, 61-67 (1984);Wood et al., Exp. Hematol., 26, 523-533 (1998)), dogs (Haurie et al.,Exp. Hematol. 27, 1139-1148 (1999); Abkowitz et al., Exp. Hematol. 16,941-945 (1988)) and in humans (Abrahamsen et al., Eur. J. Haematol. 58,333-345 (1997); Baudoux et al., Bone Marrow Transplant 22 (Suppl. 1) S12 (1998); Carulli et al., Hematologica 85, 447-448 (2000)). Similarly,cells of the gut mucosa (Schering et al., Anat. Rec. 191, 479-486(1978)), Stenn & Paus, Exp. Dermatol. 8, 229-233 (1999); Zanello et al.,J. Invest. Dermatol. 115, 757-760 (2000)) and the corneal epithelium ofthe eye (Schening et al., Anat. Rec. 191, 479-486 (1978)) exhibitcircadian organization. For human bone marrow and gastrointestinaltissues, for example, S-phase DNA synthesis preferentially occurs in themorning hours rather than in the evening or nighttime hours. Thisimplies cytotoxic agents might be less toxic and exhibit high efficacyif given at a time when the proliferative status of the cells is at anadir in these tissues.

For toxicity testing, large numbers of comparative samples are needed,thereby making the enumeration of manual CFAs for this purposeimpractical. CFAs also suffer from a lack of standardized colonyenumeration procedures, and the subjectivity and high degree ofexpertise of the personnel and the time required for accurateenumeration of the colonies. The long culture periods required tovisualize the proliferative potential of different cell populations isalso a disadvantage. However, the culture period is an inherent propertyof the cell population and cannot be changed.

Conventional cell proliferation assays have measured either ³H-thymidineor 5-bromo-deoxyuridine (BrdU) incorporation. The BrdU assay can usemicroscopy, flow cytometry or absorbance. Colorimetric tetrazoliumcompounds, in particular3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT),(Mosmann, J. Immunol. Meth. 65, 666 (1983)), have also been used.Horowitz and King, J. Immunol. Meth. 244, 49-58 (2000)) developed amulti-well, murine colony-forming assay in soft agar whereby theenumeration of cell proliferation or inhibition was measured using theMTT calorimetric method. Results were equivalent to the colony-formingassay. The number of target cells was reduced to 1.25×10⁴ cells/ml, butonly studied granulocyte/macrophage progenitor cells were tested and notstem cells, erythropoietic or megakaryopoietic progenitor cells.However, it is also desirable to have an assay system that canaccommodate the complete range of target cell populations that can becultured and subjected to drug-induced hemotoxicity effects.

Hematological malignancies rank 5th and 6th in the cause of deaths formen and women respectively and use of stem cell transplants usingperipheral blood, bone marrow and umbilical cord blood have increaseddramatically. Reconstitution of the patient after a transplant, however,usually occurs in about 14 days, which is the same time required for theconventional, manual, CFA to detect the growth potential of transplantedcells. Therefore, the usefulness of the GM-CFC assay as an indicator andquality control measure for the growth potential of the transplantablecells is limited. Reliance is often placed on measuring the number ofCD34⁺ cells by flow cytometery, even though this provides no informationas to the cell growth potential. Therefore, there is a need for asensitive, rapid and cost-effective assay that can be used as anindicator for hematopoietic engraftment and reconstitution potential.The patient would benefit significantly because, if engraftment andreconstitution of the lympho-hematopoietic system does not occur aftertransplantation, the physician can rapidly detect this rejection andproceed with a second transplant, offering reduced financialimplications in lower hospitalization and medication costs and improvedpatient comfort and recovery.

These and other objectives and advantages of the invention will becomefully apparent from the description and claims that follow or may belearned by the practice of the invention.

SUMMARY OF THE INVENTION

Briefly described, the present invention relates generally tohigh-throughput assay methods that determine the proliferative status ofhematopoietic stem and progenitor cells. The present invention furtherrelates to high-throughput assays for screening compounds that modulatethe growth of hematopoietic stem and progenitor cells and foridentifying subpopulations thereof that are suitable fortransplantation. The assay of the present invention is particularlyuseful for quality control and monitoring of the growth potential in thestem cell transplant setting and would provide improved control over thereconstitution phase of transplanted cells.

The present invention addresses the need for rapid assays that willdetermine the proliferative status of isolated hematopoietic stem andprogenitor cells and of subpopulations of differentiated cells thereof.

One aspect of the present invention provides a high-throughput assaymethod useful for rapidly determining the proliferative status of apopulation of primitive hematopoietic cells as a function of the ATPcontent of the cells, the method comprising incubating a primitivehematopoietic cell population in a cell growth medium having aconcentration of fetal bovine serum of between 0% and about 30%, aconcentration of methyl cellulose between about 0.4% and about 0.7%, andin an atmosphere having less than about 7.5% oxygen. The cell populationis contacted with a reagent capable of generating luminescence in thepresence of ATP and the luminescence generated by the reagent isdetected. The level of luminescence indicates the amount of ATP in thecell population, wherein the amount of ATP indicates the proliferativestatus of the primitive hematopoietic cells.

One embodiment of the method of the present invention further comprisesthe step of contacting the primitive hematopoietic cell population withat least one cytokine and optionally may further comprising the step ofgenerating a cell population substantially enriched in hematopoieticstem cells.

In another embodiment of the method of the present invention, the cellpopulation is substantially enriched in at least one hematopoieticprogenitor cell lineage.

Another aspect of the present invention is a high-throughput assaymethod for rapidly identifying a population of primitive hematopoieticcells having a proliferative status suitable for transplantation into apatient. The method of the present invention, therefore, may compriseincubating a primitive hematopoietic cell population in a cell growthmedium having a concentration of fetal bovine serum between 0% and about30%, a concentration of methyl cellulose between about 0.4% and about0.7%, and in an atmosphere having less than about 7.5% oxygen. Theprimitive hematopoietic cell population is contacted with at least onecytokine, typically before the incubation of the cells. Thereafter, thecell population is contacted with a reagent capable of generatingluminescence in the presence of ATP. The luminescence thereby generatedindicates the proliferative status of the primitive hematopoietic cells,which in turn indicates the suitability of the cell population fortransplantation into a recipient patient.

Yet another aspect of the present invention is a high-throughput assaymethod for rapidly identifying a compound capable of modulating theproliferative status of a population of primitive hematopoietic cells.In this aspect of the present invention, a first target cell populationcomprising primitive hematopoietic cells is incubated in cell a growthmedium having a concentration of fetal bovine serum between 0% and about30%, a concentration of methyl cellulose between about 0.4% and about0.7%, and in an atmosphere having less than about 7.5% oxygen. Themethod further comprises providing a plurality of second targetprimitive hematopoietic cell populations, contacting the first andsecond primitive hematopoietic cell populations with at least onecytokine before incubating the cell cultures, contacting the first andsecond target cell populations with at least one test compound,contacting the target cell populations with a reagent capable ofgenerating luminescence in the presence of ATP. The luminescencegenerated is detected by the reagent contacting the target cellpopulations, the level of luminescence indicating the proliferativestatus of the primitive hematopoietic cells. The proliferative status ofthe plurality of the second target cell populations with theproliferative status of the first target population of primitivehematopoietic cells not in contact with the test compound, therebyidentifying a test compound capable of modulating the proliferativestatus of a target cell population.

Additional objects and aspects of the present invention will become moreapparent upon review of the detailed description set forth below whentaken in conjunction with the accompanying figures, which are brieflydescribed as follows.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-4B illustrate the correlation between the initial plated cellconcentration (0.25, 0.5, 0.75, 1, 1.5 2×10⁵/well) and the mean (FIGS.1A, 2A, 3A and 4A respectively) or sum (FIGS. 1B, 2B, 3B, and 4Brespectively) of relative luminescence units (RLU) measured at 4 days(FIGS. 1A and 1B), 7 days (FIGS. 2A and 2B), 10 days (FIGS. 3A and 3B)and 14 days (FIGS. 4A and 4B) after culture initiation, as a function ofthe integration time and/or gain of the plate reader. In FIGS. 1A-4B thevalue 2000 represents an integration time of 2000 ms. “Max” representsthe maximum integration time. The values 200, 215, 225 or 250 representthe gains that were used with the respective integration times.

FIGS. 5A-5C illustrate histograms showing the number of cell clusterscounted manually per well and the relative luminescence units (RLU) perwell at day 7 (FIG. 5A), day 10 (FIG. 5B) and day 14 (FIG. 5C) ofincubation.

FIGS. 6A-6C graphically illustrate the lack of correlation between cellcluster counts per well and the relative luminescence units (RLU) perwell on day 7 (FIG. 6A), day 10 (FIG. 6B) and day 14 (FIG. 6C) ofculture incubation.

FIGS. 7A-7C show the direct correlation between the sum, or mean, of thecell cluster counts with the sum or mean of the relative luminescenceunits (RLU) measured on day 7 (FIG. 7A), day 10 (FIG. 7B) and day 14(FIG. 7C) of culture incubation.

FIGS. 8A-8C show the correlation between cell concentration, sum of thereplicate cell clusters and mean of the replicate cell clusters on day 7(FIG. 8A), day 10 (FIG. 8B) and day 14 (FIG. 8C) of culture incubation.

FIGS. 9A-9C show the correlation between the original manual 4-wellassay and the 96-well assay, method of the present invention. Theresults were plotted as either the sum or mean of the replicatesobtained on day 7 (FIG. 9A), day 10 (FIG. 9B) and day 14 (FIG. 9C) ofculture.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A full and enabling disclosure of the present invention, including thebest mode known to the inventor of carrying out the invention, is setforth more particularly in the remainder of the specification, includingreference to the Examples. This description is made for the purpose ofillustrating the general principles of the invention and should not betaken in the limiting sense.

The methods of the present invention provides high-throughput assays fordetecting and measuring the proliferative status of populations ofprimitive hematopoietic stem and progenitor cells, and cell lineagesderived therefrom.

The methods of the present invention are especially useful when appliedto populations of primitive hematopoietic cells including primary cellsisolated from peripheral blood cells and bone marrow cells andhematopoietic stem and progenitor cells. The methods of the presentinvention, however, may be applied to any population of proliferatingcells, including cells isolated from tissues and solid tumors.

The methods of the present invention can also be used to distinguishsubpopulations of cells that may differ in the response to cytotoxicinhibitors, or activators such as cytokines The methods may be used tooptimize the inhibitors to achieve maximum efficacy against asubpopulation of proliferating cells. An optimized dose, determined froman isolated small sample of the cell population of a patient, may beadministered to the proliferating cells in vivo, wherein the optimizeddose may be administered systemically to the human or animal patienthaving the proliferating subpopulation of cells, thereby reducing thelikelihood of potentially harmful side-effects to the recipient patient.

The high-throughput assay methods of the present invention may also beused to determine the proliferative status of a population ofhematopoietic stem or progenitor cells to determine their suitabilityand acceptability for transplantation into a recipient animal or humanpatient.

DEFINITIONS

The term “animal” as used herein refers to any vertebrate animal otherthan a human having a population of cells wherein at least onesubpopulation of the cells may be proliferating or induced toproliferate. The term “animal” as used herein also refers to mammalsincluding, but not limited to, bovine, ovine, porcine, equine, canine,feline species, non-human primates including apes and monkies, rodentssuch as rat and mouse, and lagomorphs such as rabbit and hare.

The term “tissue” as used herein refers to a group or collection ofsimilar cells and their intercellular matrix that act together in theperformance of a particular function. The primary tissues areepithelial, connective (including blood), skeletal, muscular, glandularand nervous.

The term “cell” or “cells” as used herein refers to any cell populationof a solid or non-solid tissue including, but not limited to, aperipheral blood cell population, bone marrow cell population, aleukemic cell line population and a primary leukemic cell linepopulation or a blood stem cell population. The cells may behematopoietic cells, including bone marrow, umbilical cord blood, fetalliver cells, yolk sac and differentiating embryonic stem cells ordifferentiating primordial germ cells or embryonic germ cells. The cellsmay be a primary cell line population including, but not limited to, aleukemic cell line. Examples of leukemic cell lines include, but are notlimited to, an acute lymphocytic leukemia, an acute myeloid leukemia, achronic lymphocytic leukemia, a chronic myeloid leukemia and a pre-Bacute lymphocytic leukemia. Such cell lines include, but are not limitedto, acute myelogenous leukemia, acute T-cell leukemia, acutelymphoblastic leukemia, chronic myeloid leukemia, acute monocyticleukemia and B-cell leukemia. The term “target cell population” as usedherein refers to any cell population, especially hematopoietic stem andprogenitor cells, or subpopulations thereof, that may be contacted witha test compound, wherein the test compound may modulate theproliferation of the cells in a positive or a negative directiondepending upon the compound and the target cell population.

The term “cell line” refers to cells that are harvested from a human oranimal adult or fetal tissue, including blood and cultured in vitro,including primary cell lines, finite cell lines, continuous cell lines,and transformed cell lines.

The term “cell lineage” as used herein refers to a cell line derivedfrom a progenitor or stem cell, including, but not limited to ahematopoietic stem or progenitor cell.

The term “cell cycle” as used herein refers to the cycle of stages inthe replication of a eukaryotic cell. The cycle comprises the fourstages G1, S, G2 and M, wherein the S phase is that portion of the cyclewherein the nucleic acid of the cell is replicated. Thus, a cellidentified as being in the S-phase of the cell cycle is also identifiedas being a proliferating cell.

The term “proliferative status” as used herein refers to whether apopulation of hematopoietic stem or progenitor cells, or a subpopulationthereof, are dividing and thereby increasing in number, in the quiescentstate, or whether the cells are not proliferating, dying or undergoingapoptosis.

The terms “modulating the proliferative status” or ‘modulating theproliferation” as used herein refers to the ability of a compound toalter the proliferation rate of a population of hematopoietic stem orprogenitor cells A compound may be toxic wherein the proliferation ofthe cells is slowed or halted, or the proliferation may be enhanced suchas, for example, by the addition to the cells of a cytokine or growthfactor.

The term “quiescent” refers to cells that are not actively proliferatingby means of the mitotic cell cycle. Quiescent cells (which include cellsin which quiescence has been induced as well as those cells which arenaturally quiescent, such as certain fully differentiated cells) aregenerally regarded as not being in any of the four phases G1, S, G2 andM of the cell cycle; they are usually described as being in a G0 state,so as to indicate that they would not normally progress through thecycle. Cultured cells can be induced to enter the quiescent state byvarious methods including chemical treatments, nutrient deprivation,growth inhibition or manipulation of gene expression, and induced toexit therefrom by contacting the cells with cytokines or growth factors.

The term “primary cell” refers to cells obtained directly from a humanor animal adult or fetal tissue, including blood. The “primary cells” or“cell lines” may also be derived from a solid tumor or tissue, that mayor may not include a hematopoietic cell population, and can be suspendedin a support medium. The primary cells may comprise a primary cell line.

The term “primitive hematopoietic cell” as used herein refers to anystem, progenitor or precursor cell that may be induced to differentiateand/or proliferate to form a population of hematopoietic cells.

The term “hematopoietic stem cells” as used herein refers to pluripotentstem cells or lymphoid or myeloid stem cells that, upon exposure to anappropriate cytokine or plurality of cytokines, may either differentiateinto a progenitor cell of a lymphoid or myeloid cell lineage orproliferate as a stem cell population without further differentiationhaving been initiated. “Hematopoietic stem cells” include, but are notlimited to, colony-forming cell-blast (CFC-blast), high proliferativepotential colony forming cell (HPP-CFC) and colony-formingunit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) cells.

The terms “progenitor” and “progenitor cell” as used herein refer toprimitive hematopoietic cells that have differentiated to adevelopmental stage that, when the cells are further exposed to acytokine or a group of cytokines, will differentiate further to ahematopoietic cell lineage. “Progenitors” and “progenitor cells” as usedherein also include “precursor” cells that are derived from some typesof progenitor cells and are the immediate precursor cells of some maturedifferentiated hematopoietic cells. The terms “progenitor”, and“progenitor cell” as used herein include, but are not limited to,granulocyte-macrophage colony-forming cell (GM-CFC), megakaryocytecolony-forming cell (CFC-mega), burst-forming unit erythroid (BFU-E),colony-forming cell-megakaryocyte (CFC-Mega), B cell colony-forming cell(B-CFC) and T cell colony-forming cell (T-CFC). Precursor cells”include, but are not limited to, colony-forming unit-erythroid (CFU-E),granulocyte colony forming cell (G-CFC), colony-forming cell-basophil(CFC-Bas), colony-forming cell-eosinophil (CFC-Eo) and macrophagecolony-forming cell (M-CFC) cells.

The term “cytokine” as used herein refers to any cytokine or growthfactor that can induce the differentiation of a hematopoietic stem cellto a hematopoietic progenitor or precursor cell and/or induce theproliferation thereof. Suitable cytokines for use in the presentinvention include, but are not limited to, erythropoietin,granulocyte-macrophage colony stimulating factor, granulocyte colonystimulating factor, macrophage colony stimulating factor,thrombopoietin, stem cell factor, interleukin-1, interleukin-2,interleukin-3, interleukin-6, interleukin-7, interleukin-15, Flt3L,leukemia inhibitory factor, insulin-like growth factor, and insulin. Theterm “cytokine” as used herein further refers to any natural cytokine orgrowth factor as isolated from an animal or human tissue, and anyfragment or derivative thereof that retains biological activity of theoriginal parent cytokine. The cytokine or growth factor may further be arecombinant cytokine or a growth factor such as, for example,recombinant insulin. The term “cytokine” as used herein further includesspecies-specific cytokines that while belonging to a structurally andfunctionally related group of cytokines, will have biological activityrestricted to one animal species or group of taxonomically relatedspecies, or have reduced biological effect in other species.

The terms “cell surface antigen” and “cell surface marker” as usedherein may be any antigenic structure on the surface of a cell. The cellsurface antigen may be, but is not limited to, a tumor associatedantigen, a growth factor receptor, a viral-encoded surface-expressedantigen, an antigen encoded by an oncogene product, a surface epitope, amembrane protein which mediates a classical or atypical multi-drugresistance, an antigen which mediates a tumorigenic phenotype, anantigen which mediates a metastatic phenotype, an antigen whichsuppresses a tumorigenic phenotype, an antigen which suppresses ametastatic phenotype, an antigen which is recognized by a specificimmunological effector cell such as a T-cell, and an antigen that isrecognized by a non-specific immunological effector cell such as amacrophage cell or a natural killer cell. Examples of “cell surfaceantigens” within the scope of the present invention include, but are notlimited to, CD3, CD4, CD8, CD34, CD90 (Thy-1) antigen, CD117, CD38,CD56, CD61, CD41, glycophorin A and HLA-DR, AC133 defining a subset ofCD34⁺ cells, CD19, and HLA-DR. Cell surface molecules may also includecarbohydrates, proteins, lipoproteins or any other molecules orcombinations thereof, that may be detected by selectively binding to aligand or labeled molecule and by methods such as, but not limited to,flow cytometry.

The term “cell surface indicator” as used herein refers to a compound ora plurality of compounds that will bind to a cell surface antigendirectly or indirectly, and thereby selectively indicate the presence ofthe cell surface antigen. Suitable “cell surface indicators” include,but are not limited to, cell surface antigen-specific monoclonal orpolyclonal antibodies, or derivatives or combinations thereof, and whichmay be directly or indirectly linked to a signaling moiety. The “cellsurface indicator” may be a ligand that can bind to the cell surfaceantigen, wherein the ligand may be a protein, peptide, carbohydrate,lipid or nucleic acid that is directly or indirectly linked to asignaling moiety.

The term “flow cytometer” as used herein refers to any device that willirradiate a particle suspended in a fluid medium with light at a firstwavelength, and is capable of detecting a light at the same or adifferent wavelength, wherein the detected light indicates the presenceof a cell or an indicator thereon. The “flow cytometer” may be coupledto a cell sorter that is capable of isolating the particle or cell fromother particles or cells not emitting the second light.

The term “reagent capable of generating luminescence in the presence ofATP” as used herein refers to a single reagent or combination ofcomponents that, in the presence of ATP, will generate luminescence. Theamount of luminescence may be reliably related to the amount of ATPpresent. An example of a reagent suitable for use in the presentinvention is the combination of luciferin and luciferase as described byCrouch et al. (J. Immunol. Meth. 160, 81-88 (2000)) and Bradbury et al.(J. Immunol. Meth. 240, 79-92 (2000) incorporated herein by reference intheir entireties.

The term “toxicity” as used herein refers to the ability of a compoundor a combination of compounds to negatively modulate the proliferationof a population of hematopoietic stem or progenitor cells. It will beunderstood that the toxicity of a compound or compounds may be effectiveagainst one hematopoietic cell lineage and not against another, and mayfurther include the ability of a compound to modulate thedifferentiation of a hematopoietic stem or progenitor cell.

The term “differentially distinguishable” as used herein refers tohematopoietic stem and progenitor cells, or any other animal cell, theproliferation status of which may be usefully determined by the assaymethods of the present invention and which can be characterized intosubpopulations based on, for example, different complements of cellsurface markers.

Following longstanding law convention, the terms “a” and “an” as usedherein, including the claims, are understood to mean “one” or “more”.

ABBREVIATIONS

Abbreviations used in the present specification include the following:IL, interleukin; PBMC, peripheral blood mononuclear cells; PBS,phosphate-buffered saline (10 mM phosphate, 138 mM NaCl, 2.7 mM KCl, pH7.4); FBS, fetal bovine serum; BSA, bovine serum albumen

Reference now will be made in detail to the aspects and embodiments ofthe invention. Each example is provided by way of explanation of theinvention, and not a limitation of the invention. In fact, it will beapparent to those skilled in the art that various modifications,combination, additions, deletions and variations can be made in thepresent invention without departing from the scope or spirit of theinvention. For instance, features illustrated or described as part ofone embodiment can be used in another embodiment to yield a stillfurther embodiment. It is intended that the present invention coverssuch modifications, combinations, additions, deletions and variations ascome within the scope of the appended claims and their equivalents.

The high-throughput assay methods of the present invention comprise thedetection and enumeration of a population of hematopoietic cells bymeasuring the metabolic activity of samples of proliferating cells asindicated by their ATP content. The ATP content can be measured bydetecting the luminescence generated by a ATP-dependent reactionrequiring, for example, contacting the cells with an ATP-releasing agentand an ATP-monitoring agent. A suitable system for detecting ATP by theemission of luminescence comprises the combination of luciferin andluciferase, although it is contemplated that any method that will emit adetectable signal, the intensity of which may be correlated to theamount of ATP in a cell culture may be within the scope of the presentinvention.

The high-throughput assay method of the present invention allows for thedetection of actively proliferating subpopulations of hematopoietic stemand progenitor cell lineages that have been induced to undergoproliferation by exposure of the cell population to one or morecytokines. Most hematopoietic cell lineages can be induced toproliferate by contacting the cell population with at least onecytokine. It is, therefore, contemplated that a cytokine, or combinationof cytokines, may be selected to induce the proliferation of a selectedcell lineage. It is further contemplated to be within the scope of theassay methods of the present invention for a plurality of primitivehematopoietic stem or progenitor cell populations to be contacted with aplurality of cytokines or combinations of cytokines, therebyestablishing populations of different proliferating cell lineages. Thevarious proliferating cell lineages may then be used as target cells andcontacted with one or more test compounds. The cell proliferationmodulating activities, including toxicity, of the compounds orcombinations and/or doses thereof may be compared and contrasted, aswell as how the various cell lineages will react to the test compounds.

High-Throughput Assays of Hematopoietic Stem and Progenitor CellProliferation

Primitive hematopoietic cells can be isolated from suitable animal orhuman tissues including, for example, peripheral blood, bone marrow, orumbilical cord blood. Mononuclear cells, for example peripheral bloodmononuclear cells (PBMCs) may be further isolated by methods such asdensity-gradient centrifugation. It is contemplated to be within thescope of the present invention for the primitive cell population to befurther subdivided into isolated subpopulations of cells that arecharacterized by specific cell surface markers. The methods of thepresent invention may further include the separation of cellsubpopulations by methods such as high-speed high-speed cell sorting,typically coupled with flow cytometry.

For example, the channels of a flow-cytometer and high-speed cell sortercould be set at 530 nm, typically used for FITC labeling, 670 nm usedfor APC labeling, and a UV channel, for Hoechst (Ho) 33342 or DAPIstaining. Fluorescent compensation software such as the System II orExpo 32 (Beckman Coulter) can allow full use of all of these channels.Cell subpopulations can be selected based on the presence or absence ofcell membrane antigen markers, the intracellular pH, and the cell cyclestatus. Exemplary methods for selectively distinguishing subpopulationsof hematopoietic cells are described, for example, in PCT applicationSerial No: 20010012620, incorporated herein in by reference in itsentirety.

Multiparameter analysis may be conducted on primary normal and leukemicsamples or leukemic cell lines. The methods of the present invention,however, may be applied or adapted to any non-leukemic hematopoieticstem or progenitor cell population that might include a subpopulation ofproliferating cells. An antigen indicator conjugated to APC can be usedto selectively detect a normal blood stem cell subpopulation. Aliquotsof cells may be labeled with panels comprising more than one biomarker.An example of one such panel incorporates CD38-FITC, CD34-APC, SNARF andHo33342. Other examples of possible panels can include substitutingCD38-FITC with CD117(c-kit)-FITC, with CD91 (Thy-1)-FITC or withAC133-FITC.

The procedures of the present invention, therefore, can providetechniques to analyze combinations of cell markers as described above,or those specific for other lympho-hematopoietic lineages todifferentiate the effects of inhibitors on normal different cellsubpopulations. A similar reasoning can be applied to leukemic cellpopulations that also show aberrant flow cytometric profilesdistinguishable from the normal population. A typical example would bechronic myeloid leukemia in chronic phase. However, in the case of ALL,the leukemic cell population can be defined by a high proportion ofCD19⁺ cells. Therefore, CD19 is a biomarker that can be used todifferentiate between leukemic and non-leukemic populations. Theselected cell subpopulations can then be applied to the high-throughputassays of the present invention, as described in Examples 1 and 2 below.

Cell surface indicators may be contacted with the hematopoietic stem orprogenitor cells or leukemic cells thereof and the varioussubpopulations may be selectively separated by techniques such as flowcytometry or by attaching the cell surface indicators directly orindirectly to a separable solid support such as magnetic beads. Thebeads and the attached cells thereon can be isolated by a magneticfield.

In the methods of the present invention as described, for example, inExamples 1 and 2 below, target hematopoietic stem and/or progenitorcells are isolated from animal or human tissues and suspended at cellconcentrations ranging from about 1-5×10² to about 1-2×10⁵/ml. Sincetypical assay volumes are 100 μl, actual cell concentrations in theassay test vessels will be diluted to 1/10 of the original starting cellconcentration. The cells are mixed and suspended in methyl cellulosecontaining 0% to about 30% concentration of fetal bovine serum (FBS), 1%detoxified bovine serum albumin (BSA), iron-saturated human transferrinat a fmal concentration of 1×10⁻¹⁰ mol/L, α-thioglycerol at a finalconcentration of 1×10⁻⁴ mol/L and cytokines/growth factors. The methylcellulose concentration in the assays of the present invention isbetween about 0.4% and about 0.7%, with a preferred concentration formost cell populations of about 0.7%. One exemplary medium is Iscove'sModified Dulbecco's Medium (IMDM, Life Technologies, Rockville, Md.)although other suitable media capable of supporting the growth ofhematopoietic cells may also be used. Low fetal bovine serumconcentrations of between 0% and 10% can also be used. When the assaymethods of the present invention are used under serum-free conditions,insulin (10 μg/ml) and, where necessary, low density lipoproteins (40μg/ml) can replace the FBS.

A stock cell culture is aliquoted into sample chambers. While samplechambers may be the wells of a multi-well tissue culture plate, such asa 48- or 96-well plate, it is also contemplated to be within the scopeof the present invention to conduct the assays of the present inventionin any other suitable reaction vessels including, but not limited to,individual tubes, wells of plates and the like. Culture plates with awell surface area of about 35 mm² and a low ring of about 2 mm high areespecially useful and allow colonies to be counted that are against thewall of the ring. Preferably the sample chambers are not tissue culturetreated. For luminescence assays to be performed, multi-well plates thatreduce background light emission or scatter when the plates are beingenumerated in the plate reader may also be used. While it is desirableto use replicate reactions, it is to be understood that a singlereaction sample may be used for determining the proliferative status ofcells for each data point. However, replicate reactions are to bepreferred wherever an increase in accuracy is necessary. For example,reactions may be replicated once, twice or more times, including on asingle multi-well plate, although quadruple reactions are preferred.

The assay methods of the present invention especially contemplate thatthe cultures can be incubated in a humidified atmosphere having a lowoxygen tension for a period preferably extending to about 10 days butalso to at least about 14 days. A suitable oxygen concentration range isfrom about 3.5% oxygen to about 7.5% oxygen, most preferably about 5.0%oxygen, and further comprising about 5% CO₂ as described by Bradley etal. (J. Cell Physiol. 97, 517-522 (1968) and Rich & Kubanek (Exp. Hemat.52, 579-588 (1982) incorporated herein by reference in their entireties.

Regardless of the instrument parameters used, there is a directcorrelation between the cell concentration plated in the wells and themean, or sum, of the relative luminescence units obtained. To avoidlarge standard deviations, an integration time of 1000 ms may be used,although other integration times may usefully be selected.

The high-throughput assay method of the present invention furtherincludes contacting a hematopoietic stem or progenitor cell populationwith at least one cytokine that can induce the proliferation of the stemor progenitor cell population. It is contemplated that the cytokine of acombination of cytokines may be selected to induce the differentiationand proliferation of selected subpopulations of hematopoietic celllineages. Exemplary cytokines include, but are not limited toerythropoietin, granulocyte-macrophage colony stimulating factor,granulocyte colony stimulating factor, macrophage colony stimulatingfactor, thrombopoietin, stem cell factor, interleukin-1, interleukin-2,interleukin-3, interleukin-6, interleukin-7, interleukin-15, Flt3L,leukemia inhibitory factor. Additional growth factors may also beincluded to boost the proliferative status of a particular culture ofcells including such factors as insulin-like growth factor, insulin andrecombinant insulin. Examples of cytokines or combinations thereof thatmay be used in the assay methods of the present invention and thespecific targeted stem, progenitor or precursor cell types, and theresulting expanded cell lineages are given in Example 3 and Table 1below.

High-Throughput Assay Methods for Toxicity Testing with HematopoieticStem and Progenitor Cells

It is further contemplated that a cell lineage that is induced toproliferate by contacting a first primitive hematopoietic stem orprogenitor cell population with a cytokine or combination of cytokinesmay further be contacted with a test compound that may have a cytotoxiceffect or a cell proliferation enhancing effect. The degree ofmodulation of cell proliferation or differentiation may also bedetermined by comparing the proliferation of the cell lineage in thepresence of the test compound, and in its absence from the culture of asecond targeted cell population or plurality of second cell populations.It is within the scope of the assay methods of the present invention fora plurality of test compounds to be compared for their cytotoxic effectson one, or a plurality, of proliferating target cell lineages. To theseends, a plurality of hematopoietic stem or progenitor cell populationsmay, for example, be plated in the wells of a multi-well plate or inindividual chambers, thereby allowing rapid testing of multiple samples.

It is also contemplated that the high-throughput assays of the presentinvention may be used to determine the ability of a test compound toincrease the proliferation of a population of hematopoietic stem orprogenitor cells. Such proliferation enhancing compounds include, forexample, cytokines and growth factors.

It is further contemplated that the assay methods of the presentinvention may be used with a range of concentrations of the testcompound which may be contacted with a plurality of cell populations ofthe same cell lineage, whereupon the IC50 or the IC90 for the testcompound acting against the targeted cell population or a subpopulationthereof may be calculated.

High-Throughput Assay Methods for Screening Hematopoietic Stem andProgenitor Cell Populations for Suitability for Transplantation

The high-throughput assay methods of the present invention are alsosuitable for screening a population of hematopoietic stem or progenitorcells to determine the proliferation status of the cells orsubpopulations thereof wherein the proliferative status will indicatethe suitability of the stem or progenitor cells for transplantation intoa recipient animal or human host. The high-throughput assay of thepresent invention will allow the selection of populations of primitivehematopoietic cell that will likely proliferate and maintain engrafmentwithin the recipient patient.

By determining the sum or mean of the relative luminescence units (RLU)in all replicates of a single sample at a specified time point duringthe incubation procedure, for example at 4, 7, 10 or 14 days ofincubation, the assay can be used to rapidly and quantitativelydetermine: (a) the proliferative status of a hematopoietic stem orprogenitor cell population or of cells of a specific progenitor anddifferentiation lineage and compare such in parallel assays; (b) ifcells from a particular source exhibit a normal or abnormalproliferative capacity; and (c) whether a compound (e.g. growth factor,cytokine, drug, neutraceutical, environmental agent), will have apositive or negative effect of the proliferative status of the cells ina particular cell population. The assay, even of multiple samples, canbe completed within 30 min, calculated from the time of adding the ATPreleasing agent to the conclusion of the luminescence measurement.

The high-throughput stem/progenitor cell assay (HT-SPCA) of the presentinvention does not count colonies or differentiate between colony types.Rather, the HT-SPCA of the present invention measures the proliferationstatus of cells within the colonies by determining the amount of ATPbeing produced by the cells. With colony growth in the methyl celluloseassay system of the present invention, some cells in the cultures willbegin to proliferate and form aggregates or clusters. However, theproliferative status of the cell population may be limited due to theirlate stage of differentiation. Thus, a small colony may ensue within ashort incubation period, but cell proliferation will rapidly cease.

In the assay methods of the present invention, the culture conditionsinclude α-thioglycerol to maintain molecules in a reduced form, and thecultures are incubated under low oxygen tension of between about 3.5%oxygen and about 7.5% oxygen, both conditions reducing oxygen toxicity.The cell aggregate or colony can be maintained in a stagnant ornon-proliferative state for between about 2 and about 3 weeks. Othercells, however, that are developmentally more primitive, for example,stem and progenitor cells, have a greater proliferative capacity andwill begin to form colonies after a certain lag period of time. Thesecells will continue to divide throughout the whole of the incubationperiod. Eventually, the proliferative capacity of the cells within thesecolonies will also decrease and finally cease.

This ability of the assay method of the present invention to distinguishprimitive hematopoietic cells from more mature, differentiated lineagescontrasts with the conventional manual assay methods. In the manualassay, in which colonies are counted under a microscope, proliferatingcells cannot be readily distinguished from non-proliferating cells. Thesize of the colony, however, may indicate the “primitiveness” of thecell that gave rise to that colony. Thus, the larger the colony, thegreater the possibility of the colony deriving from a more primitivecell. In the HT-SPCA method of the present invention, the size of thecolony of the present invention, however, is irrelevant. Rather, it isthe proliferative status of the cells within the colonies within thesame well that is measured, as documented in Example 3 below.

One aspect of the present invention, therefore, is a high-throughputassay method for rapidly determining the proliferative status of apopulation of primitive hematopoietic cells, the method comprising thesteps of providing a cell population comprising primitive hematopoieticcells, incubating the cell population in a cell growth medium comprisinga concentration of fetal bovine serum between 0% and about 30% and aconcentration of methyl cellulose between about 0.4% and about 0.7%, andin an atmosphere having between about 3.5% oxygen about 5.5% oxygen,preferably 5.0% oxygen, contacting the cell population with a reagentcapable of generating luminescence in the presence of ATP, and detectingluminescence generated by the reagent contacting the cell population,the level of luminescence indicating the amount of ATP in the cellpopulation, wherein the amount of ATP indicates the proliferative statusof the primitive hematopoietic cells.

In one embodiment of the method of the present invention, theconcentration of fetal bovine serum is between about 0% and 10%.

In another embodiment of the method of the present invention, theconcentration of methyl cellulose is about 0.7%.

In yet another embodiment of the present invention, the concentration ofoxygen in the atmosphere is about 5%.

Another embodiment of the method of the present invention furthercomprises the step of contacting the primitive hematopoietic cellpopulation with at least one cytokine and optionally may furthercomprise the step of generating a cell population substantially enrichedin hematopoietic stem cells.

One embodiment of the method of the present invention comprises the stepof generating a cell population substantially enriched in at least onehematopoietic progenitor cell lineage.

In one embodiment of the method of the present invention, the primitivehematopoietic cells are hematopoietic stem cells.

In another embodiment of the method of the present invention, theprimitive hematopoietic cells are hematopoietic progenitor cells.

In yet another embodiment of the method of the present invention, thepopulation of primitive hematopoietic cells comprises hematopoietic stemcells and hematopoietic progenitor cells.

In still another embodiment of the method of the present invention, theprimitive hematopoietic cells are primary hematopoietic cells.

In one embodiment of the method of the present invention, the primaryhematopoietic cells are isolated from animal tissue selected from thegroup consisting of peripheral blood, bone marrow, umbilical cord blood,yolk sac, fetal liver and spleen.

In one embodiment of the method of the present invention, the animaltissue is obtained from a human.

In one embodiment of the method of the present invention, the animaltissue is selected from bone marrow, yolk sac, fetal liver and spleen.

In various embodiments of the method of the present invention, theanimal is a mammal.

In various embodiments of the method of the present invention, themammal is selected from the group consisting of cow, sheep, pig, horse,goat, dog, cat, non-human primates, rodents, rabbit and hare.

In another embodiment of the method of the present invention, the animaltissue is human tissue further selected from the group consisting ofperipheral blood, bone marrow, umbilical cord blood fetal liver andspleen.

In yet another embodiment of the method of the present invention, theprimary hematopoietic stem cells are isolated from peripheral blood.

Still another embodiment of the method of the present invention furthercomprises the step of selecting a differentially distinguishablesubpopulation of primitive hematopoietic cells from the population ofprimitive hematopoietic cells, wherein the subpopulation of cells isdefined by cell surface markers thereon.

In one embodiment of the method of the present invention, the step ofselecting a differentially distinguishable subpopulation of primitivehematopoietic cells from the population of primitive hematopoietic cellscomprises the steps of contacting the population of primitivehematopoietic cells with at least one cell surface marker indicatorcapable of selectively binding to a cell surface marker of adifferentially distinguishable subpopulation of cells, and selectivelyisolating the at least one subpopulation of cells binding the at leastone indicator.

In one embodiment of the method of the present invention, the cellsurface marker is selected from the group consisting of CD3, CD4, CD8,CD34, CD90 (Thy-1) antigen, CD117, CD38, CD56, CD61, CD41, glycophorinA, HLA-DR, AC133 defining a subset of CD34⁺ cells, CD19, and HLA-DR.

In one embodiment of the method of the present invention, the cellsurface marker is CD34⁺.

In one embodiment of the method of the present invention, thesubpopulation of differentially distinguishable primitive cells isselectively isolated by magnetic bead separation.

In another embodiment of the method of the present invention, thesubpopulation of differentially distinguishable primitive cells isselectively isolated by flow cytometry and cell sorting.

In yet another embodiment of the method of the present invention, thepopulation of primitive hematopoietic cells comprises at least one stemcell lineage selected from the group consisting of colony-formingcell-blast (CFC-blast), high proliferative potential colony forming cell(HPP-CFC) colony-forming unit-granulocyte, erythroid, macrophage,megakaryocyte (CFU-GEMM).

In the various embodiments of the methods of the present invention, thepopulation of primitive hematopoietic cells comprises at least onehematopoietic progenitor cell lineage selected from the group consistingof granulocyte-macrophage colony-forming cell (GM-CFC), megakaryocytecolony-forming cell (CFC-mega), macrophage colony-forming cell (M-CFC),granulocyte colony forming cell (G-CFC), burst-forming unit erythroid(BFU-E), colony-forming unit-erythroid (CFU-E), colony-formingcell-basophil (CFC-Bas), colony-forming cell-eosinophil (CFC-Eo),colony-forming cell-megakaryocyte (CFC-Mega), B cell colony-forming cell(B-CFC) and T cell colony-forming cell (T-CFC).

Also, in the various embodiments of the methods of the presentinvention, the reagent capable of generating luminescence in thepresence of ATP comprises luciferin and luciferase.

Also, in the various embodiments of the methods of the presentinvention, the at least one cytokine is selected from the groupconsisting of erythropoietin, granulocyte-macrophage colony stimulatingfactor, granulocyte colony stimulating factor, macrophage colonystimulating factor, thrombopoietin, stem cell factor, interleukin-1,interleukin-2, interleukin-3, interleukin-6, interleukin-7,interleukin-15, Flt3L, leukemia inhibitory factor, insulin-like growthfactor, and insulin.

In one embodiment of the method of the present invention, the at leastone cytokine is stem cell factor, interleukin-7 and Flt3L, and whereinthe at least one cytokine generates a cell population substantiallyenriched in colony-forming cells blast (CFC-Blast) stem cells.

In another embodiment of the method of the present invention, the atleast one cytokine is macrophage colony stimulating factor,interleukin-1, interleukin-3, interleukin-6 and stem cell factor, andwherein the at least one cytokine generates a cell populationsubstantially enriched in hematopoietic high proliferative potentialcolony-forming cell (HPP-CFC) stem cells.

In yet another embodiment of the method of the present invention, the atleast one cytokine is erythropoietin, granulocyte-macrophage colonystimulating factor, granulocyte colony stimulating factor, stem cellfactor, interleukin-3, interleukin-6, and optionally Flt3L, and whereinthe at least one cytokine generates a cell population substantiallyenriched in hematopoietic colony-forming cell erythroid, macrophage,megakaryocyte (CFC-GEMM) stem cells.

In still another embodiment of the method of the present invention, theat least one cytokine is selected from the group consisting oferythropoietin, erythropoietin and interleukin-3, erythropoietin andstem cell factor and erythropoietin, stem cell factor and interleukin-3,and wherein the at least one cytokine generates a cell populationsubstantially enriched in the hematopoietic burst forming unit-erythroid(BFU-E) progenitor cells.

In still yet embodiment of the method of the present invention, the atleast one cytokine is further selected from granulocyte-macrophagecolony stimulating factor, granulocyte-macrophage colony stimulatingfactor and interleukin-3, and granulocyte-macrophage colony stimulatingfactor, interleukin-3 and stem cell factor, and wherein the at least onecytokine generates a cell population substantially enriched inhematopoietic granulocyte-macrophage colony-forming cell (GM-CFC)progenitor cells.

In another embodiment of the method of the present invention, the atleast one cytokine is further selected from the groups consisting ofthrombopoietin, and thrombopoietin, interleukin-3 and interleukin-6, andwherein the at least one cytokine generates a cell populationsubstantially enriched in the hematopoietic megakaryocyte colony-formingcell (CFC-Mega) progenitor cells.

In yet another embodiment of the method of the present invention, the atleast one cytokine is further selected from interleukin-2, andinterleukin-7, Flt3L and interleukin-15, and wherein the at least onecytokine generates a cell population substantially enriched in thehematopoietic T cell colony forming cell (T-CFC) progenitor cells.

In still another embodiment of the method of the present invention, theat least one cytokine is selected from the group consisting ofinterleukin-7, and interleukin-7 and Flt3L, and wherein the at least onecytokine generates a cell population substantially enriched in thehematopoietic B cell colony-forming cell (B-CFC) progenitor cells.

In still yet another embodiment of the method of the present invention,the at least one cytokine is erythropoietin and wherein the at least onecytokine generates a cell population substantially enriched in thehematopoietic colony-forming unit-erythroid (CFU-E) progenitor cells.

In another embodiment of the method of the present invention, the atleast one cytokine is selected from the group consisting ofgranulocyte-colony stimulating factor and granulocyte-macrophage colonystimulating factor, and wherein the at least one cytokine generates acell population substantially enriched in the hematopoietic granulocytecolony-forming cell (G-CFC) progenitor cells.

In yet another embodiment of the method of the present invention, the atleast one cytokine is selected from the group consisting ofinterleukin-3, and interleukin-3 and stem cell factor, and wherein theat least one cytokine generates a cell population substantially enrichedin the hematopoietic colony-forming cell-Basophil (CFC-Bas) progenitorcells.

In still another embodiment of the method of the present invention, theat least one cytokine granulocyte-macrophage colony stimulating factor,interleukin-3 and interleukin-5, and wherein the at least one cytokinegenerates a cell population substantially enriched in the hematopoieticcolony-forming cell-eosinophil (CFC-Eo) progenitor cells.

In still yet another embodiment of the method of the present invention,the at least one cytokine is selected from the group consisting ofmacrophage colony stimulating factor, macrophage colony stimulatingfactor and granulocyte-macrophage colony stimulating factor andinterleukin-7, and granulocyte-macrophage colony stimulating factor, andwherein the at least one cytokine generates a cell populationsubstantially enriched in the hematopoietic macrophage colony-formingcell (M-CFC) progenitor cells.

One embodiment of the method of the present invention further comprisesthe step of identifying a population of primitive hematopoietic cellshaving a proliferative status suitable for transplantation into arecipient patient.

Another embodiment of the method of the present invention furthercomprises the steps of providing a population of primitive hematopoieticcells comprising a target cell population, contacting the target cellpopulation with a test compound, and determining the ability of the testcompound to modulate the proliferative status of the target cellpopulation.

In one embodiment of the method of the present invention, the populationof primitive hematopoietic cells comprises a plurality of target cellpopulations, and the method further comprises the steps of contactingthe plurality of target cell populations with at least one testcompound, determining the ability of the at least one test compound toalter the proliferation of the target cell population by comparing theproliferative status of the plurality of target cell populations withthe proliferative status of a target population of primitivehematopoietic cells not in contact with the test compound, andidentifying the at least one test compound modulating the proliferativestatus of a target cell population.

Another aspect of the present invention, therefore, is a high-throughputassay method for rapidly identifying a population of primitivehematopoietic cells having a proliferative status suitable fortransplantation into a patient, comprising the steps providing a cellpopulation comprising primitive hematopoietic cells, incubating the cellpopulation in cell a growth medium comprising between 0% and 30% fetalbovine serum and a concentration of methyl cellulose between about 0.4%and about 0.7%, and in an atmosphere having between about 3.5% and about7.5% oxygen, contacting the primitive hematopoietic cell population withat least one cytokine selected from the group consisting oferythropoietin, granulocyte-macrophage colony stimulating factor,granulocyte colony stimulating factor, macrophage colony stimulatingfactor, thrombopoietin, stem cell factor, interleukin-1, interleukin-2,interleukin-3, interleukin-6, interleukin-7, interleukin-15, Flt3L,leukemia inhibitory factor, insulin-like growth factor, and insulin,contacting the cell population with a reagent capable of generatingluminescence in the presence of ATP, and detecting luminescencegenerated by the reagent contacting the at least two cell populations,the level of luminescence indicating the proliferative status of theprimitive hematopoietic cells, and wherein the proliferative status ofthe primitive hematopoietic cells indicates the suitability of the cellpopulation for transplantation into a recipient patient.

In one embodiment of this aspect of the method of the present invention,contacting the population of primitive hematopoietic cells with at leastone cytokines generates a cell population substantially enriched in ahematopoietic stem cell lineage.

In one embodiment of the method of the present invention, thehematopoietic stem cell lineage is selected from the group consisting ofcolony-forming cell-blast (CFC-blast), high proliferative potentialcolony forming cell (HPP-CFC) colony-forming unit-granulocyte,erythroid, macrophage, megakaryocyte (CFU-GEMM).

In another embodiment of this aspect of the method of the presentinvention, contacting the population of primitive hematopoietic cellswith at least one cytokine generates a cell population substantiallyenriched in at least one hematopoietic progenitor cell lineage.

In the various embodiments of this aspect of the method of the presentinvention, the population of primitive hematopoietic cells comprises atleast one hematopoietic progenitor cell lineage selected from the groupconsisting of granulocyte-macrophage colony-forming cell (GM-CFC),megakaryocyte colony-forming cell (CFC-mega), macrophage colony-formingcell (M-CFC), granulocyte colony forming cell (G-CFC), burst-formingunit erythroid (BFU-E), colony-forming unit-erythroid (CFU-E),colony-forming cell-basophil (CFC-Bas), colony-forming cell-eosinophil(CFC-Eo), colony-forming cell-megakaryocyte (CFC-Mega), B cellcolony-forming cell (B-CFC) and T cell colony-forming cell (T-CFC).

Yet another aspect of the present invention is a high-throughput assaymethod for rapidly identifying a compound capable of modulating theproliferative status of a population of primitive hematopoietic cells,comprising the steps of providing a first target cell populationcomprising primitive hematopoietic cells, incubating the cell populationin cell a growth medium comprising between 0% and 30% fetal bovine serumand a concentration of methyl cellulose between about 0.4% and about0.7%, and in an atmosphere having between about 3.5% and about 7.5%oxygen, providing a plurality of second target cell populationscomprising primitive hematopoietic cells, contacting the first andsecond target primitive hematopoietic cell populations with at least onecytokine selected from the group consisting of erythropoietin,granulocyte-macrophage colony stimulating factor, granulocyte colonystimulating factor, macrophage colony stimulating factor,thrombopoietin, stem cell factor, interleukin-1, interleukin-2,interleukin-3, interleukin-6, interleukin-7, interleukin-15, Flt3L,leukemia inhibitory factor, insulin-like growth factor, and insulin,contacting the first and second target cell populations with at leastone test compound, contacting the first and second target cellpopulations with a reagent capable of generating luminescence in thepresence of ATP, detecting luminescence generated by the reagentcontacting the first and second target cell populations, the level ofluminescence indicating the proliferative status of the primitivehematopoietic cells, and comparing the proliferative status of theplurality of the second target cell populations with the proliferativestatus of the first target population of primitive hematopoietic cellsnot in contact with the test compound, thereby identifying a testcompound capable of modulating the proliferative status of a target cellpopulation.

In the various embodiments of this aspect of the method of the presentinvention, contacting the first and second target populations ofprimitive hematopoietic cells with at least one cytokine generates cellpopulations substantially enriched in hematopoietic stem cells.

In other embodiments of the methods of the present invention, thehematopoietic stem cells are selected from the group consisting ofcolony-forming cell-blast (CFC-blast), high proliferative potentialcolony forming cell (HPP-CFC) colony-forming unit-granulocyte,erythroid, macrophage, megakaryocyte (CFU-GEMM).

Also, in the various embodiments of this aspect of the method of thepresent invention, contacting the first and second target populations ofprimitive hematopoietic cells with at least one cytokine generates cellpopulations substantially enriched in at least one hematopoieticprogenitor cell lineage.

In the various embodiments of this aspect of the method of the presentinvention, the at least one hematopoietic progenitor cell lineageselected from the group consisting of granulocyte-macrophagecolony-forming cell (GM-CFC), megakaryocyte colony-forming cell(CFC-mega), macrophage colony-forming cell (M-CFC), granulocyte colonyforming cell (G-CFC), burst-forming unit erythroid (BFU-E),colony-forming unit-erythroid (CFU-E), colony-forming cell-basophil(CFC-Bas), colony-forming cell-eosinophil (CFC-Eo), colony-formingcell-megakaryocyte (CFC-Mega), B cell colony-forming cell (B-CFC) and Tcell colony-forming cell (T-CFC).

One embodiment of the method of the present invention further comprisesthe steps of contacting a target cell population with at least twoconcentrations of a test compound, and calculating the IC50 of the testcompound.

Another embodiment of the method of the present invention furthercomprises the steps of contacting a target cell population with at leasttwo concentrations of a test compound and calculating the IC90 of thetest compound.

The present invention is further illustrated by the following examples,which are provided by way of illustration and should not be construed aslimiting. The contents of all references, published patents and patentscited throughout the present application are hereby incorporated byreference in their entirety.

Example 1 Preparation of Incubated Hematopoietic Stem or ProgenitorCells Isolation of Mononuclear Cells

Mononuclear cells (MNC) were prepared from human peripheral blood, bonemarrow or umbilical cord blood by density gradient centrifugation onFicoll-Paque Plus by diluting the cell suspension 1:1 with sterile PBSand transferring up to 30 ml to a 15-20 ml cushion of Ficoll.Centrifugation was at 400 g for 20 mins at room temperature. Thesupernatant was discarded and the cells were resuspended in 50 ml ofsterile PBS and re-centrifuged at 200 g for 10 mins at room temperature.Thereafter, the supernatant was discarded and the cells were resuspendedin IMDM to ensure a single cell suspension. A cell count was determined.If not used immediately, the cells were placed on ice or at 4° C.

Cells from peripheral blood mononucleocytes (MNCs) were prepared at afinal concentration of 2×10⁶ cells/ml. MNCs from bone marrow andumbilical cord blood were prepared at final concentrations of 0.5-1×10⁶and 0.5-1×10⁵ respectively.

Bulk Reagent Solutions

The following components were mixed in sterile tubes, usually in thefollowing order, so that the final total volume of the culture mixturewas 600 μl or multiples thereof. The volume prepared depended on thenumber of replicate assays required. A “master mix” of 600 μl wassufficient for 4-5 replicates of 100 μl each. If volumes of the mastermix greater than 600 μl were required, medium was added first followedby methyl cellulose. This mixture was then mixed on a vortex mixer. Iflarge quantities of methyl cellulose were added first, it becamedifficult to mix the components adequately.

(a) Serum-Containing Cultures

-   -   (i) Methyl cellulose (stock at 2.6% v/v),. 160 μl, (final        concentration, 0.7% v/v)    -   (ii) Fetal bovine serum (FBS), 180 μl    -   (iii) α thioglycerol, 6 μl at a final concentration of 1×10⁻⁴ M    -   (iv) Human or bovine iron-saturated transferrin, 6 μl, final        concentration of 1×10⁻¹⁰ g/ml    -   (v) Growth factors, individually or in combination, were        selected from the following: erythropoietin, 1-3 U/ml;        granulocyte-macrophage colony stimulating actor, 10-20 ng/ml;        granulocyte colony stimulating factor, 10-20 ng/ml; macrophage        colony stimulating factor, 10-20 ng/ml; thrombopoietin, 50        ng/ml; stem cell factor, 50 ng/ml; interleukin-1, 10-20 ng/ml;        interleukin-2, 2-10 ng/ml; interleukin-3, 20 ng/ml;        interleukin-6, 20 ng/ml; interleukin-7, 10 ng/ml. The volume        added depended upon the concentration of the cytokine/growth        factor stock solution, but typically were not greater than 6-10        μl. All growth factors were diluted in IMDM containing either 5%        FBS or 1% BSA    -   (vi) Cells diluted in IMDM to the required final concentration        as described above and added at 60 μl.    -   (vii) IMDM added to give a final stock solution volume of 600 μl        (or multiples thereof).        (b) Serum-Free Cultures.

For serum-free conditions, the fetal bovine serum of (ii) above wasreplaced by a mixture of bovine serum albumen, transferrin and insulinadded as a preformed mixture (BIT 9500, Stem Cells Technologies,Vancouver).

Once the basic components were added, including the growth factors thatwere required for the specific cell type to be analyzed, the contentswere vortexed to yield a homogenous mixture. The reaction mixes wereleft to stand for a few minutes before dispensing to the wells of a96-well plate or other arrays of receptacles. When cultures wereincubated for more than 7 days, the outer wells of a 96-well plate werefilled with 100 μl of sterile water to ensure that the cultures did notdesiccate, even when a humidified tissue culture incubator was used.

Using a 1 ml syringe with an 18 gauge 1.5″ needle, or a repeater pipettewith a syringe capable of dispensing aliquots of 100 μl,serum-containing, or a serum-free culture prepared as described abovewas withdrawn slowly and dispensed into each of the replicate wells of a96-well plate while ensuring that little or none of the master mixtouched the sides of the wells.

Sample Incubation

Once the samples had been dispensed into the wells, the 96-well platewas placed in a fully humidified tissue culture incubator at 37° C. Thecells were incubated in a low oxygen tension atmosphere of 5% CO₂ and 5%oxygen (obtained by replacing the oxygen in the incubator with nitrogengas). The incubation period depended on the cell population to betested.

Example 2 Measurement of the ATP Content of Incubated Hematopoietic Stemor Progenitor Cells

After the incubation time has elapsed, the reagents from the ViaLightHS™ kits (Lumitech) were prepared for use. If necessary, the number ofcell clusters (aggregates) or colonies that had developed in the wellsof the incubated 96-well plates could be counted under an invertedmicroscope to ensure that a correlation between the sum, or mean, of theratio of clusters/colonies to the relative luminescence units (RLU) wasobtained (see below).

All reagents were allowed to attain room temperature before use. The ATPmonitoring reagent was reconstituted as described by the manufacturersby adding 10 ml of the supplied buffer to the lyophilized reagent andwaiting 15 mins. Alternatively, 1 ml of the buffer was used toreconstitute the reagent and the latter was then aliquoted into 1.5 mlmicrotubes and frozen while protected from light. Aliquots were thenthawed and diluted to 1 ml final volumes using the supplied buffer asneeded. The ATP monitoring reagent was protected from light at alltimes.

The required quantity of ATP releasing reagent was transferred into thereagent trough and 100 μl aliquots were transferred, using a multi-tippipette, to each row or column of wells of the 96-well plated previouslyincubated as described in Example 1 above. After dispensing the reagentto one row or column, the contents of the wells were mixed at least 4-5times with the pipette, so that the reagents mixed well with the methylcellulose master mixes. Addition of the reagents diluted the methylcellulose and mixing ensured that the cells came into contact with theATP releasing reagent. This step had to be performed in a similar mannerfor all wells.

Once the ATP releasing reagent had been dispensed into all of the wellscontaining incubated cultures, and mixed therein, the plates weretypically incubated in the dark for 5 min, although the incubation couldproceed for up to 30 min without loss of sensitivity.

The required amount of ATP monitoring reagent was transferred to a new,clean trough and 20 μl of the reagent pipetted into each of the wellswhile ensuring that the contents of each well was mixed thoroughly. Theplates were immediately transferred to a plate reader and theluminescence measured using an integration time of 1000 ms.

Measurement was given as relative luminescence units (RLU). In addition,since no two makes of luminescence readers are the same, the gainrequired to obtain sufficient RLU by each machine had to be empiricallydetermined, but could also be standardized between machines as follows:

If space allowed on the same plate as the wells containing the incubatedcell cultures, 100 μl of a 10⁻⁶ M standard ATP diluted in IMDM from a10⁻⁵ M ATP stock solution was added to each of 4-6 wells. 100 μl of ATPreleasing agent was then added and incubated for the same period as hadbeen used for the cell culture wells. 20 μl of ATP monitoring reagentwas then added and the plate transferred to the plate reader and theluminescence measured at an integration time of 1000 ms. The gain wasadjusted such that the luminescence from the ATP standard wasapproximately 20,000 RLU. By adjusting the gain of the machine to obtainthis number of RLU, and then reading the remaining wells of the plate(containing the incubated cells), no overflow values occurred, therebyobviating the need for a second or multiple reading. In all cases, theluminescence was measured in the shortest possible time period possible,because the luminescence decreased rapidly with time.

The correlation between the initial plated cell concentration (0.25,0.5, 0.75, 1, 1.5 2×10⁵/well) and the mean (FIGS. 1A, 2A, 3A and 4Arespectively) or sum (FIGS. 1B, 2B, 3B, and 4B respectively) of relativeluminescence units (RLU) measured at 4 days (FIGS. 1A and 1B), 7 days(FIGS. 2A and 2B), 10 days (FIGS. 3A and 3B) and 14 days (FIGS. 4A and4B) after culture initiation, as a function of the integration timeand/or gain of the plate reader. In FIGS. 1A-4B the value 2000represents an integration time of 2000 ms. “Max” represents the maximumintegration time. The values 200, 215, 225 or 250 represent the gainsthat were used with the respective integration times.

Example 3 Hematopoietic Stem and Progenitor Cell Lines and TheirAssociated Cytokine Effectors

Hematopoietic stem and progenotor cells are induced to differentiateinto hematopoietic cell subpopulations by exposure to one or more growthfactors/cytokines, as shown in Table 1 below.

TABLE 1 Development Population Stimulatory Growth stage LineagePopulation name abbreviation factors and cytokines Stem cell NoneLong-term culture- LTC-IC Stimulated by (Most initiating cellsmicroenvironmental primitive in cells vitro stem cell) Stem cell NoneColony-forming cell- CFC-Blast Flt3L, SCF and IL-7 (Very Blast primitivein vitro stem cell) Stem cell None High proliferative HPP-CFC IL-1,IL-3, IL-6, SCF, (Primitive in potential colony- M-CSF vitro stemforming cell cell) Stem cell None Colony-forming cell CFC-GEMM IL-3,IL-6, GM-CFC, G- (Most mature granulocyte, CSF, EPO, and SCF in vitrostem erythroid and/or Flt3L cell) macrophage, megakaryocyte ProgenitorErythroid Burst-forming unit- BFU-E EPO Erythroid IL-3 and EPO SCF andEPO IL-3, SCF and EPO Progenitor Granulocyte- Granulocyte- GM-CFC GM-CSFMacrophage macrophage colony- IL-3 and GM-CSF forming unit IL-3, SCF andGM-CSF Progenitor Megakaryocyte Megakaryocyte CFC-Mega TPOcolony-forming cell IL-3, IL-6 and TPO Progenitor T T cellcolony-forming T-CFC IL-2 lymphocyte cell IL-7, Flt3L and IL-15Progenitor B B cell colony- B-CFC IL-7 lymphocyte forming cell IL-7 andFlt3L Precursor Erythroid Colony-forming cell- CFU-E EPO erythroidPrecursor Myeloid - Granulocyte colony- G-CFC G-CSF Neutrohil formingcell GM-CSF, high concentrations Precursor Myeloid - Colony-formingcell- CFC-Bas IL-3 Basophil basophil IL-3 and SCF Precursor Myeloid -Colony-forming CFC-Eo GM-CSF and IL-5 and Eosinophil cells-eosinophilIL-3 Precursor Macrophage Macrophage colony- M-CFC M-CSF forming cellM-CSF, GM-CSF, IL-3 GM-CSF, low concentrations

Example 4 Proliferation of Hematopoietic Stem and Progenitor CellsMeasured by Colony Counting and ATP Determination

When cell proliferation was measured as a function of time in culture,some aggregates or colonies contained cells that were proliferating,while others were not, as shown in FIGS. 5A-5C. Wells, therefore, couldcontain few colonies, but still exhibit high cell proliferation. Theresults shown in FIGS. 5A-5C show that the number of cell clusterscounted per well does not correlate with the cell proliferation asdetected using the luminescence of the present invention.

In contrast, in those wells in which minimal or no cluster formation wasdetected, luminescence could be detected. In some wells, theluminescence was significantly greater than expected from the number ofcell clusters counted, indicating that cell proliferation was occurringand that the proliferating cells, were primitive because of theirincreased proliferative capacity. On day 10, most wells contained cellsthat were proliferating. By day 14, this proliferative capacity was onlyseen in some wells, indicating that proliferation has ceased (RLU lowerthan the cell cluster count) or is declining. Those wells exhibiting asignificantly greater RLU than determined by manual cell clustercounting showed that cells were present that were capable of extensiveproliferation and were probably stem cells.

Little or no correlation existed between the number of individualcolonies and the luminescence, as shown in FIGS. 6A-6C. However, underthe typical incubation and culture conditions of the assay of thepresent invention, a well containing a few colonies, but with highindicated cell proliferation, showed that the cells in the culture havehigh proliferative capacity. In other words, the cells were primitive innature.

The number of colonies in a well did not generally correlate with theRLU from that well, as shown in FIGS. 6A-6C. In contrast, however, inthe high-throughput assay method of the present invention, theluminescence measurements were made over the whole area of each andevery replicate well, and not from the individual cell aggregates orcolonies within these areas. The sum of the luminescent values ofaggregates or colonies, or the mean of the aggregates or colonies fromall replicate wells, can be predicted to correlate with the sum or meanof the luminescence emitted from the replicate wells, as shown in FIGS.7A-7C.

There was a direct correlation between the sum, or mean, of all the cellaggregates or colonies from all replicate wells and the sum or mean ofthe RLU from all replicate wells. Furthermore, this relationship wascell dose dependent, as illustrated in FIGS. 8A-8C.

These results, as opposed to those in FIGS. 6A-6C indicate that usingthe sum or the mean of the replicates from particular sample, in thiscase, replicates at different cell concentrations, a direct correlationexists between the 3 parameters. If measurement of luminescence isdepicted as the sum or the mean of the replicates, there was also be adirect correlation with the sum and/or mean of the cell clusters, asshown in FIGS. 8A-8D.

To validate the 96-well plate assay, experiments using both assaysystems were performed in parallel. A direct correlation exists betweenthe 4-well and the 96-well plate assay, as shown in FIGS. 9A-9C andindicates that the results obtained from the latter have been validated.

Example 5 Use of High-Throughput Stem/Progenitor Cell Assays toDetermine the Ability of a Test Compound to Modulate the Proliferationof Hematopoietic Stem and Progenitor Cells

The HT-SPCA of the present invention is used to test dose responses fora variety of compounds that can interact with hematopoietic stem andprogenitor cells. The agents either stimulate or inhibit and/or killhematopoietic cells. Increasing doses of an agent can stimulate cells,but then be inhibitory by being toxic and causing necrosis. Other agentscan be toxic at high doses, but induce apoptosis at lowerconcentrations.

Agents with known action on hematopoietic stem and progenitor cellsinclude, 5-fluorouracil (5-FU), hydroxyurea, cytosine arabinoside(ara-C). busulphan, 3′azido-3′deoxythymide (AZT), cycloheximide,actinomycin D, etoposide, BCNU, doxorubicin, cisplatin (lowhemotoxicity) and carboplatin. Growth factors known to inhibit theproliferation of stem and progenitor cells such as interferon-γ (IFNγ),tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGFβ)are also tested. Neutraceuticals include, for example, theanti-inflammatory phytochemicals, black and green tea polyphenols,resveritrol, limonene and curcumin.

For these and other agents to be tested, mononuclear cells derived fromperipheral blood, bone marrow and cord blood are used. CD34+ cellsderived from these tissues can also be used. HT-SPCA assays forCFU-GEMM, GM-CFC, BFU-E, CFC-Mega and CFU-E, CFC-blast, HPP-CFC, M-CFCand G-CFC induced to proliferate and differentiate by contacting thecell populations with the appropriate cytokine of combinations ofcytokines as given in Example 3, Table 1, above, are also performed.

HT-SPCA can be used to detect and predict hemotoxicity againsthematopoietic stem and progenitor cell populations. To validate theinhibition/hemotoxicity of the agents, both manual CFA and the HT-SPCAare performed in parallel. One of the end points is to determine theIC50 and IC90 for the drug.

1. A high-throughput assay method for rapidly identifying a populationof primitive hematopoietic cells having a proliferative status suitablefor transplantation into a patient, comprising the steps of: (a)providing a cell population comprising primitive hematopoietic cells;(b) incubating the primitive hematopoietic cell population in a cellgrowth medium comprising fetal bovine serum having a concentration ofbetween 0% and about 30%, methyl cellulose having a concentration ofbetween about 0.4% and about 0.7%, and in an atmosphere having betweenabout 3.5% oxygen and 7.5% oxygen; (c) contacting the primitivehematopoietic cell population with a proliferation agent, theproliferation agent comprising one or more growth factors, one or morecytokines, or combinations thereof; (d) contacting the cell populationwith a reagent capable of reacting with ATP and generating luminescencein the presence of ATP; and (e) detecting luminescence generated by thereagent that reacted with the ATP in the primitive hematopoietic cellpopulation, the level of luminescence indicating the amount of ATP inthe primitive hematopoietic cell population, wherein the amount of ATPindicates the proliferative status of the primitive hematopoietic cellsand wherein the proliferative status of the primitive hematopoieticcells indicates the suitability of the cell population fortransplantation into a recipient patient.
 2. The method of claim 1,wherein contacting the population of primitive hematopoietic cells witha proliferation agent generates a cell population substantially enrichedin a hematopoietic stem cell lineage.
 3. The method of claim 2, whereinthe hematopoietic stem cell lineage is selected from the groupconsisting of colony-forming cell-blast (CFC-blast), high proliferativepotential colony forming cell (HPP-CFC), colony-formingunit-granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM), andlong-term culture-initiating cell (LTC-JC).
 4. The method of claim 1,wherein contacting the population of primitive hematopoietic cells witha proliferation agent generates a cell population substantially enrichedin at least one hematopoietic progenitor cell lineage.
 5. The method ofclaim 4, wherein the at least one hematopoietic progenitor cell lineageis selected from the group consisting of granulocyte-macrophagecolony-forming cell (GM-CFC), megakaryocyte colony-forming cell(CFC-mega), macrophage colony-forming cell (M-CFC), granulocyte colonyforming cell (G-CFC), burst-forming unit erythroid (BFU-E),colony-forming unit-erythroid (CFU-E), colony-forming cell-basophil(CFC-Bas), colony-forming cell-eosinophil (CFC-Eo), colony-formingcell-megakaryocyte (CFC-Mega), B cell colony-forming cell (B-CFC), and Tcell colony-forming cell (T-CFC).
 6. The method of claim 1 wherein theproliferation agent is selected from the group consisting oferythropoietin, granulocyte-macrophage colony stimulating factor,granulocyte colony stimulating factor, macrophage colony stimulatingfactor, thrombopoietin, stem cell factor, interleukin-1, interleukin-2,interleukin-3, interleukin-6, interleukin-7, interleukin-15, Flt3L,leukemia inhibitory factor, insulin-like growth factor, insulin, andcombinations thereof.
 7. The method of claim 1, wherein theconcentration of fetal bovine serum is between about 0% and 10%.
 8. Themethod of claim 1, wherein the concentration of methyl cellulose isabout 0.7%.
 9. The method of claim 1, wherein the concentration ofoxygen in the atmosphere is about 5%.
 10. The method of claim 1, whereinthe reagent capable of reacting with ATP and generating luminescence inthe presence of ATP comprises luciferin and luciferase.
 11. The methodof claim 10, wherein the population of primitive hematopoietic cells isisolated from an animal tissue selected from the group consisting ofperipheral blood, bone marrow, umbilical cord blood, yolk sac, fetalliver, and spleen.
 12. The method of claim 1, wherein the proliferationagent comprises erythropoietin, granulocyte-macrophage colonystimulating factor, granulocyte colony stimulating factor, stem cellfactor, interleukin-3, interleukin-6, and optionally Flt3L, and whereinthe proliferation agent generates a cell population substantiallyenriched in hematopoietic colony-forming unit-granulocyte, erythroid,macrophage, megakaryocyte (CFU-GEMM) stem cells.
 13. The method of claim1, wherein the proliferation agent comprises stem cell factor,interleukin-7, and Flt3L, and wherein the proliferation agent generatesa cell population substantially enriched in colony-forming cells blast(CFC-Blast) stem cells.
 14. The method of claim 1, wherein theproliferation agent comprises macrophage colony stimulating factor,interleukin-1, interleukin-3, interleukin-6, and stem cell factor, andwherein the proliferation agent generates a cell populationsubstantially enriched in hematopoietic high proliferative potentialcolony-forming cell (HPP-CFC) stem cells.
 15. The method of claim 1,wherein the proliferation agent is selected from the group consisting oferythropoietin, erythropoietin and interleukin-3, erythropoietin andstem cell factor and erythropoietin, stem cell factor and interleukin-3,and wherein the proliferation agent generates a cell populationsubstantially enriched in the hematopoietic burst forming unit-erythroid(BFU-E) progenitor cells.
 16. The method of claim 1, wherein theproliferation agent is selected from the group consisting ofgranulocyte-macrophage colony stimulating factor, granulocyte-macrophagecolony stimulating factor and interleukin-3, and granulocyte-macrophagecolony stimulating factor, interleukin-3 and stem cell factor, andwherein the proliferation agent generates a cell populationsubstantially enriched in hematopoietic granulocyte-macrophagecolony-forming cell (GM-CFC) progenitor cells.
 17. The method of claim1, wherein the proliferation agent is selected from the group consistingof thrombopoietin, and thrombopoietin, interleukin-3 and interleukin-6,and wherein the proliferation agent generates a cell populationsubstantially enriched in the hematopoietic megakaryocyte colony-formingcell (CFC-Mega) progenitor cells.
 18. The method of claim 1, wherein theproliferation agent is selected from the group consisting ofinterleukin-2, and interleukin-7, Flt3L and interleukin-15, and whereinthe proliferation agent generates a cell population substantiallyenriched in the hematopoietic T cell colony forming cell (T-CFC)progenitor cells.
 19. The method of claim 1, wherein the proliferationagent is selected from the group consisting of interleukin-7, andinterleukin-7 and Flt3L, and wherein the proliferation agent generates acell population substantially enriched in the hematopoietic B cellcolony-forming cell (B-CFC) progenitor cells.
 20. The method of claim 1,wherein the proliferation agent comprises erythropoietin and wherein theproliferation agent generates a cell population substantially enrichedin the hematopoietic colony-forming unit-erythroid (CFU-E) progenitorcells.
 21. The method of claim 1, wherein the proliferation agent isselected from the group consisting of granulocyte-colony stimulatingfactor and granulocyte-macrophage colony stimulating factor, and whereinthe proliferation agent generates a cell population substantiallyenriched in the hematopoietic granulocyte colony-forming cell (G-CFC)progenitor cells.
 22. The method of claim 1, wherein the proliferationagent is selected from the group consisting of interleukin-3, andinterleukin-3 and stem cell factor, and wherein the proliferation agentgenerates a cell population substantially enriched in the hematopoieticcolony-forming cell-Basophil (CFC-Bas) progenitor cells.
 23. The methodof claim 1, wherein the proliferation agent comprisesgranulocyte-macrophage colony stimulating factor, interleukin-3 andinterleukin-5, and wherein the proliferation agent generates a cellpopulation substantially enriched in the hematopoietic colony-formingcell-eosinophil (CFC-Eo) progenitor cells.
 24. The method of claim 1,wherein the proliferation agent is selected from the group consisting ofmacrophage colony stimulating factor, macrophage colony stimulatingfactor and granulocyte-macrophage colony stimulating factor andinterleukin-7, and granulocyte-macrophage colony stimulating factor, andwherein the proliferation agent generates a cell populationsubstantially enriched in the hematopoietic macrophage colony-formingcell (M-CFC) progenitor cells.
 25. The method of claim 1, whereinbetween step (c) and step (d), the method further comprises: (c1)incubating the primitive hematopoietic cell population for a period ofabout 4 days.
 26. The method of claim 1, wherein between step (c) andstep (d), the method further comprises: (c1) incubating the primitivehematopoietic cell population for a period of about 7 days.
 27. Themethod of claim 1, wherein between step (c) and step (d), the methodfurther comprises: (c1) incubating the primitive hematopoietic cellpopulation for a period of about 10 days.
 28. The method of claim 1,wherein between step (c) and step (d), the method further comprises:(c1) incubating the primitive hematopoietic cell population for a periodof about 14 days.